Inorganic nanomaterials have attracted extensive attention as a result of their emerging properties and their potential for a multitude of applications such as electronics, catalysis, sensors, and medical diagnosis. Many of the applications for inorganic nanomaterials, for example, metallic nanomaterials especially those composed of gold and silver, have exploited their surface plasmon resonance (SPR) properties, which are sensitive to the size, shape, composition and chemical environment of the metallic nanomaterials. With the hope of discovering novel properties for future applications, various methods have been developed to synthesize inorganic nanomaterials. Based on the unique requirements of various applications, inorganic nanomaterials have been fabricated through a variety of physical and chemical methods. Many of these methods require extreme conditions such as high temperature, elevated pressure, organic solvents, caustic pH and strong reducing reagents.
The use of biological systems, inspired by naturally evolved processes, is an emerging trend in their fabrication, and it has been reported that material size, shape, and morphology can be controlled by interactions between biomolecules and inorganic materials. During the last few decades, quite a few approaches have been developed to synthesize gold nanoparticles due to their many useful applications. These chemical methods to synthesize gold nanoparticles can be placed in two categories. One utilizes citrate to reduce gold ions in aqueous solution while the other one employs strong binding ligands to transfer gold ions from water to an organic layer where the gold ions are reduced by NaBH4 in the presence of surfactants. Gold nanoparticles synthesized by these two approaches usually require further functionalization to be utilized in future applications. To avoid additional modification steps, biomolecules including amino acids, peptides and proteins have been employed to fabricate gold nanoparticles and some of these reports have shown that the morphology, size and lattice structure can be regulated by different biomolecules.
Amino acids are small biomolecules which serve as the building blocks for protein synthesis. Amino acids have been employed as simple biotemplates to prepare gold nanomaterials and play an essential role in stabilizing the nanomaterial.
It was found that by incubating various amino acids such as lysine, arginine, tryptophan, tyrosine, and aspartic acid, with tetrachloroaurate in an aqueous solution at room temperature for about 12 hours, with lysine, arginine or tryptophan spherical gold nanoparticles can be produced, while with tyrosine both spherical and rod-shape nanoparticles are formed. It was also interestingly found that with aspartic acid single crystal hexagonal and truncated triangular shaped gold nanoplates in (111) facet can be formed. It was postulated that aspartic acid can specifically interact with the (111) gold crystalline face, thereby promoting local reduction at the interaction region. Taken together, the results indicate that shape and crystal preference can be regulated by the type of amino acid used.
The synthesis of tyrosine-, glycyl-L-tyrosine-, and arginine-reduced gold nanoparticles in alkaline conditions has also been reported. The size of the tyrosine-prepared nanoparticles was larger, on average, compared to those reduced by glycyl-L-tyrosine in identical conditions. L-arginine-prepared particles exhibited a larger size and unique morphology. In addition it was found that the size of the particles could be increased by lowering the gold concentration.
Together these data indicate that the size and morphology of the resulting nanomaterials could be manipulated by using amino acids with presumed different reduction potentials and by varying gold ion concentrations. More generally, it was demonstrated that amino acids can serve as reducing agents to initiate the growth of gold nanomaterials and stabilize them in aqueous solution.
Proteins and peptides, due to their large structural and functional diversity and their ready availability, have high utility in the manipulation of materials.
Peptides are polymers of amino acids linked by peptide bonds. Depending on the number of amino acids in the peptide molecule, one can, for example, differentiate between dipeptides, tripeptides, oligopeptides and polypeptides, to name a few. Although peptides are composed of amino acids, the conditions of amino acid-based gold nanoparticle synthesis cannot be applied to peptide-based gold nanoparticle synthesis. These differences could be due to altered projection of functionality which is a result of secondary structure formation in the peptides. As these conformationally related effects, combined with the opportunity to combine functional groups with varied chemical properties, could potentially afford a degree of nuanced control not available with amino acids, methods involving peptides have been the focus of much scientific interest.
A histidine rich peptide (AHHAHHAAD) was reported to possess a high affinity for metal ions and has been utilized to mediate the growth of gold nanoparticles in aqueous solution. Gold ions were mixed with the peptide in 1:1 molar ratio and nanoparticles were formed by addition of reducing agent (sodium citrate or sodium borohydride). The UV spectrum of the particles showed the characteristic plasmon resonance peak at 524 nm, and the average diameter of the particles was 9.5 nm. Immunoassays have been conducted and antibodies fused to these histidine rich peptides were able to detect the gold nanoparticles suggesting that the peptides mediate the growth of gold nanoparticles through surface recognition. With the confirmation that these peptides act as ligands, nanoparticles were produced and functionalized efficiently in one-pot synthesis without performing ligand exchange steps.
Subsequently, histidine rich peptides were immobilized on nanotubes to mediate the growth of uniform sized gold nanocrystals. The peptide-functionalized nanotubes were mixed with organic gold complex (Cl3AuPMe3). As the gold ions were slowly trapped by the histidine peptides they were crystallized by reduction with NaBH4, This procedure resulted in highly monodisperse gold nanocrystals with an average diameter of 6 nm. The diffraction pattern of these particles indicated that the crystal was in the (111) and (220) facet. In a subsequent report, it was found that the packing density of gold nanocrystals could be manipulated by altering the pH and ion concentration, however, the diameter of gold nanoparticles was retained. Because the packing density on the nanotubes has significant influence on conductivity, this technology could be utilized as a conductivity-tunable building block in electronics and sensor devices.
Some other peptides like the Flg and A3 peptides (DYKDDDDK and AYSSGAPPMPPF, respectively), identified by phage display have been used to synthesize gold nanoparticles in HEPES buffer. Biotinylated anti-Flg antibody could successfully recognize Flg peptides which were ligated to the surface of gold nanoparticles during particle formation and the complexes were mixed with streptavidin coated quantum dots to form bio-assembled hybrid nanostructures. Thus, with the assistance of high throughput screening techniques like phage display, peptides that have high affinity to metal ions/particles can be easily selected and employed for further applications.
Proteins are essentially polypeptides usually composed of more than 100 amino acids. Although some proteins are composed of a single polypeptide chain, other proteins comprise two or more polypeptide chains that are linked by non-covalent interactions or disulfide bridges between cysteine residues. Generally, the linear length, functional diversity, and amino acid sequence of the proteins cause them to adopt thermally stable folds upon which their biological and molecular recognition activity depend. Various proteins have been directly utilized in the fabrication process of gold nanoparticles.
Bovine serum albumin (BSA) is a suitable protein candidate for the synthesis of gold nanoparticles, as it possesses many sulfur-, oxygen-, and nitrogen-containing amino acid residues all of which have a high affinity for gold ions. After mixing BSA with gold ions (AuCl4), the reducing reagent NaBH4 was added, resulting in well dispersed gold nanoparticles with an average diameter less than 2 nm.
Infrared (IR) and Raman spectroscopy indicated that the BSA backbone and the functional groups on the amino acid side chain remained intact during the reaction. The disulfide bonds in BSA, however, were broken resulting in free thiol groups available to make strong interactions with the gold nanoparticles. This study demonstrated that proteins could be conjugated to gold nanoparticles during their formation. Another protein, d-amylase was reported to reduce gold ions (AuCl4−) while maintaining its enzymatic activity. The protein was interacting with the particle surface through free thiol groups which were possibly donating electrons for the reduction. Interestingly, the active site of the enzyme which is adjacent to these implicated cysteines was not affected during the gold particle formation as enzymatic turnover could still be observed. Although many other enzymes have been screened for protein-assisted gold nanoparticle synthesis, only EcoR I could successfully produce gold nanoparticles. The only structural similarity between α-amylase and EcoR I is that both of the proteins have free cysteines which are presumably essential in the reduction of gold ions to gold nanoparticles.
Amino acids, peptides, and proteins all seem to function by surface binding of growing gold nanoparticles. The size and shape of the resulting particles depends on the affinity of this binding along with how the binding on and off rates relate to the chemical rate of the reduction of the gold. Much optimization is therefore required to produce the gold particles and their size and shape can rarely be predicted rationally from the outset. However, some proteins assemble into unique quaternary structures and therefore could serve as platforms to template gold nanoparticle formation. In these cases the resulting material could reflect the size and 3D shape of the templating protein.
It was reported to use tobacco mosaic virus (TMV), a rod shape virus 18 nm in diameter and 300 nm in length, to perform this templation by incubating with gold ions (AuCl4−) at acidic pH. After adding the reducing agent hydrazine hydrate, gold ions which bound to the TMV surface acted as nucleation sites to promote nanoparticle growth. Spherical gold nanoparticles with diameters of 8.6±3 nm densely covered the external surface of the viral capsids.
In a subsequent report, the conditions to deposit gold nanoparticles homogeneously on TMV were optimized through repeated addition of gold ions and reducing agent in aliquots in the presence of poly-L-lysine.
Biotemplate-directed syntheses have the potential to be more “green” than traditional methods due to the required mild reaction conditions such as lower temperature, near-neutral pH, and the fact that they often employ aqueous reaction solutions. Many bio-assemblies tend to have exquisite nanostructures and their components often can be manipulated easily using molecular biological techniques. Another advantage of the biotemplate approach is that new functional groups on the biomolecules could be projected onto the growing nanomaterials enabling the potential for further applications without the need of adding stabilizing agent. Moreover, the biomolecules can prevent the agglomeration of nanomaterials in high salt solutions as well as increase the solubility of nanomaterials in aqueous solutions. Proteins that assemble into nanocage structures have been reported as templates to produce many types of nanoparticles.
Ferritin is a well characterized protein that assembles into a spherical ball with a hollow interior and it is an excellent candidate for fabrication of nanoparticles due to its unique structure and high stability. The ferritin proteins assemble into robust nanoscale cages and are ubiquitously expressed in both prokaryotes and eukaryotes. The ferritin protein from horse spleen, for example, is composed of 24 subunits that form an octahedral, hollow sphere with an exterior diameter of 12 nm and an interior cavity of 7 nm. The biological function of ferritin is to sequester and mineralize Fe(O)OH inside the cavity so as to prevent cytosolic and serum iron from forming cell-destructive, reactive oxygen species. Iron is transported into the cavity through eight hydrophilic channels on the threefold symmetry axes and mineralized within the protein shell. It has been speculated that channels on the fourfold axes serve as exit pathways for cations during demineralization.
Upon removal of their mineralized cores, empty cages of ferritin (i.e., apoferritin) have been used as size-constrained reaction vessels to synthesize different types of nanoparticles including metals, oxides, hydroxides, carbonates, and semiconductors. These particles possess a narrow size distribution arising from growth restriction within the cage whose uniformity is a result of the precision of protein self-assembly. Moreover, the protein cages could enhance the solubility and chemical stability of the particles. Therefore, multiple methods have been developed to mineralize nanoparticles using ferritins.
Many of the strategies have capitalized on natural electrostatic interactions or specific binding between metal ions and the interior surface of native ferritins to increase the local concentration and thus facilitate the formation of nanoparticles. Due to the anionic nature of the ferritin cavity, and the direction of the electrostatic gradient in the ion-entry channels at the threefold axes, only cations can be successfully used with this strategy. Other strategies respond to the fact that some metal ions have a natural affinity for the ferritin exterior, or in some cases no preference for either the interior nor exterior, resulting in substantial mineralization on the outside of the ferritin. In one attempt to remedy this problem, ion-bound ferritins are first subjected to dialysis or chromatography before subsequent reduction inside the cavity. Alternatively, ammonium ions, ethylenediaminetetraacetic acid (EDTA), or polyelectrolytes have been included in the reaction solution to retard or prevent mineralization on the outside of the cage. Another strategy has been to genetically or chemically modify the proteins to endow the cavity with an enhanced ion binding affinity or the ability to promote particle formation.
The mineralization of gold nanoparticles using ferritin cages was recently reported. Reaction between either monoanionic AuCl or neutral AuCl3 and unmodified horse spleen apoferritin (HSAFn) resulted only in gold mineralization on the outside of the protein, and the size of these deposits could be controlled by the choice of the reductant. In a subsequent report, human heavy chain ferritin was modified by removing solvent-exposed gold-binding amino acids, such as cysteine and histidine, from the outer surface and by lining the interior surface with cysteine residues. Gold nanoparticles were successfully incorporated inside the cavity of this modified protein by the addition of AuCl3 followed by reduction with 3-(N-morpholino)propanesulfonic acid (MOPS).
Previous studies demonstrated that gold nanoparticles were generally generated outside the protein cages and some form(s) of modification of the proteins were required. As many different protein cages have distinct shapes and sizes, such strategies were limited to a specific protein cage or a specific group of protein cages. This means that these strategies could not potentially be used with any natural protein cage and any protein cage that is commercially available,
Thus it is an object of the present invention to address at least the problems mentioned above and to provide a universal method for generating gold nanoparticles of different shapes and sizes encased by protective, solublizing, and easily functionalizable protein.